1. Field of the Invention
The present invention relates generally to the field of biosynthetic production of 3-O-deacylated-4′-monophosphoryl lipid A (3D-MLA). More particularly, it concerns methods of improving the yield of desired 3D-MLA congeners or minimizing the cost of purifying lipopolysaccharide (LPS) precursors of 3D-MLA.
2. Description of Related Art
It has long been recognized that enterobacterial lipopolysaccharides (LPS) are potent stimulators of the immune system. A variety of responses, both beneficial and harmful, can be elicited by submicrogram amounts of LPS. The fact that some of the responses are harmful, and some of these can be fatal, has precluded clinical use of LPS per se. It has been observed that the component of LPS most responsible for endotoxic activity is lipid A.
Accordingly, much effort has been made towards attenuating the toxic attributes of LPS or lipid A without diminishing the immunostimulatory benefits of these compounds. Notable among these efforts were those of Edgar Ribi and his associates, which resulted in the production of the lipid A derivative 3-O-deacylated-4′-monophosphoryl lipid A (3D-MLA; compositions comprising 3D-MLA are commercially available under the trade name MPL® from Corixa Corporation (Seattle, Wash.)). 3D-MLA has been shown to have essentially the same immunostimulatory properties as lipid A but lower endotoxicity (Myers et al, U.S. Pat. No. 4,912,094). Myers et al. also reported a method for production of 3D-MLA, as follows. First, LPS or lipid A obtained from a deep rough mutant strain of a gram-negative bacterium (e.g. Salmonella minnesota R595) is refluxed in mineral acid solutions of moderate strength (e.g. 0.1 N HCl) for a period of approximately 30 min. This leads to dephosphorylation at position 1 of the reducing-end glucosamine and decarbohydration at the 6′ position at the non-reaming glucosamine of lipid A. Second, the dephosphorylated decarbohydrated lipid A (a.k.a. monophosphoryl lipid A or MLA) is subject to base hydrolysis by, for example, dissolving in an organic solvent such as chloroform:methanol (CM) 2:1 (v/v), saturating the solution an aqueous solution of 0.5 M Na2CO3 in pH 10.5, and flash evaporating solvent. This leads to selective removal of the β-hydroxymyristic acid moiety at position 3 of the lipid A, resulting in 3-O-deacylated-4′-monophosphoryl lipid A (3D-MLA).
The quality of the 3D-MLA produced by the above method is highly dependent on the purity and composition of the LPS obtained from the grain-negative bacterium. For one example, the lipid A component of LPS is a mixture of closely related species that contain between about 5-7 fatty acid moieties. In the formation of 3D-MLA, as is clear from the above discussion, one fatty acid moiety is removed, yielding 3D-MLA with between about 4-6 fatty acid moieties. It is generally held that 3D-MLA with at least 6 fatty acid moieties is preferred in terms of the combination of maintained or enhanced immunostimulatory benefits, reduced toxicity, and other desirable properties (Qureshi and Takayama, in “The Bacteria,” Vol. XI (Iglewski and Clark, eds.), Academic Press, 1990, pp. 319-338).
For another example, commercial scale extraction of LPS from gram-negative bacteria typically involves the Chen method (Chen et al. J. Infect. Dis. 128:543 (1973)); namely, extraction with CM, which leads to an LPS- and phospholipid-rich CM phase from which LPS can later be purified. However, purification of LPS from the LPS- and phospholipid-rich CM phase typically requires multiple precipitation steps to obtain LPS of sufficient purity for use in immunostimulatory applications such as, for example, use as a vaccine adjuvant.
Therefore, it would be desirable to have methods for conveniently preparing highly pure LPS compositions. Further, it would be desirable to have methods for generating LPS compositions which compositions have 3D-MLA with increased levels of hexaacyl congeners.
Known fermentation techniques have been used to prepare cultures of gram-negative bacteria comprising readily purifiable LPS. These known techniques typically involve harvesting or bacterial cultures at early stationary phase, in keeping with standard bacteriological practices. However, it has been observed that the degree of acylation of LPS produced according to known conditions is variable. For example, the content of heptaacyl species in the lipid A of S. minnesota R595 can vary from 20% to 80%, depending on the batch (Rietschel et al., Rev. Infect. Dis. 9:S527 (19871). This variability in heptaracyl congener content would result in the significant differences in the hexaacyl congener content in the 3D MLA prepared from these LPS batches.